Solid lipidic nanospheres suitable to a fast internalization into cells

ABSTRACT

The present invention relates to pharmaceutical compositions in form of solid lipidic nanospheres able to rapidly penetrate into the cells, comprising as an active substance a lipidic substance consisting of an ester of α-tocopherol or δ-tocopherol or of cholesterol or of glycerol with a carboxylic acid selected from acetic acid, propionic acid, butyric acid and succinic acid, useful in the treatment of tumoral pathologies and of Mediterranean anaemia.

FIELD OF THE INVENTION

The present invention relates to pharmaceutical compositions in form ofsolid lipidic nanospheres able to fast penetrate into the cells, theprocess for their preparation and their use in the treatment of tumoralpathologies and of Mediterranean anaemia.

PRIOR ART

It is known from literature that the carboxylic acids having a lownumber of carbon atoms, such as acetic acid, propionic acid, butyricacid and succinic acid, and their derivatives, may inhibit theproliferation of the tumoral cells, in particular in the case of coloncancers (H. P. Scheppach, F. Ritcher, Eur. J. Cancer Prevention, 4,373-378, 1995, and 31, 1077-1080, 1995).

There are in particular experimental proofs showing theantiproliferative activity of the butyric acid salts, for example of thesodium salt, towards a great variety of neoplastic cells (S. P. Landonet al., Cancer res., 48, 6161-6165, 1988; D. Coradini et al., CellProlif., 30, 149-159, 1997; H. Yamamoto et al., Int. J. Cancer, 76,897-902, 1998). Recent studies showed that sodium butyrate is able tomodulate the expression of the oncogenes and of the genes regulating theapoptosis in cells from different histotypes (O. C. Velasquez et al., J.Parenteral Enteral Nutr., 20, 243-250, 1996; M. Mandal, R. Kumar, CellGrowth Diff., 7, 311-318, 1996).

It is moreover known that the carboxylic acids themselves andlor certainderivatives thereof may help in a significant way, in the case of theMediterranean anaemia, the transformation of β-globin to γ-globin, orfetal globin, resulting in an improvement of the disease (S. P. Perineet al., New England J. Medicine, 328, 81-86, 1993, A. F. Collins et al.,Blood, 85, 43-49, 1995).

At present, the use of such compounds is however strongly limited by thedifficulty in reaching effective plasmatic concentrations, owing to theshort half-life time, which makes the metabolism and the excretion ofsaid substances too fast. In order to obtain satisfactory resultstherefore one ought to administrate high amounts of acid, with thedrawback of causing harmful side effects.

Therefore one feels the need to have an adequate system of release forthese substances, which allows to decrease their doses, thus minimisingthe side effects.

The document WO 94/20072 (Westesen K. at al.) describes particles ofbioactive agents wherein the matrix is constituted by the bioactiveagent itself. Substances particularly suitable for the formulation assaid particles are drugs and other bioactive materials which are poorlywater soluble. A long list of said substances, from anesthetics tovirustatics, comprising tocopherol acetate and tocopherol succinate isreported.

No information about the internalization of said particles into cells isgiven.

SUMMARY

Now the Applicant found new pharmaceutical compositions allowing toovercome the drawbacks of the prior art, showing a surprisingly highbiological activity.

Said pharmaceutical compositions are prepared in form of solid lipidicnanospheres characterised in that they comprise as an active substance alipidic substance consisting of cholesteryl butyrate and if necessaryone or more further pharmacologically active substances.

A further object of the present invention is the process for thepreparation of said lipidic nanospheres, comprising the following steps:

a) heating of a mixture comprising a lipidic substance and one or moresurfactants at a temperature such as to take the mixture to melting;

b) heating of a mixture consisting of water and one or moreco-surfactants at a temperature at least equal to the step a) one;

c) hot mixing under mild stirring of the mixture of the step b) with themixture of the step a), with the achievement of a microemulsion;

d) dispersion of the microemulsion obtained in the step c) in pre-cooledwater;

e) washing of the dispersion of the step d) with distilled water bydiafiltration;

f) freeze-drying of the product obtained in the step e) or its hotsterilisation, characterised in that said lipidic substance consists ofcholesteryl butyrate.

The pharmaceutical compositions in form of solid lipidic nanospheresobject of the present invention are useful in the treatment of all thepathological conditions for which the administration of the abovementioned carboxylic acids is effective, and they are particularlysuitable for the treatment of tumoral pathologies and of theMediterranean anaemia.

The characteristics and the advantages of the solid lipidic nanospheresas a release system for the carboxylic acids according to the presentinvention and of the related preparation process will be pointed out indetail in the following description.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to pharmaceutical compositions in form ofsolid lipidic nanospheres obtained from microemulsions of a lipidicsubstance, stabilised by at least a surfactant and by one or morecosurfactants.

With the term lipidic nanospheres in the present invention we meanparticles having an average diameter lower than 300 nm.

For the preparation of said microemulsions a lipidic substance in amixture with one or more surfactants, and if necessary one or morefurther pharmacologically active substances, is heated to melting;separately a mixture consisting of water and one or more cosurfactantsis heated to a temperature at least equal to that one at which themixture containing the lipidic substance melts. The aqueous mixture isthen hot added under mild stirring to the mixture containing the lipidicsubstance, obtaining a microemulsion.

The so obtained microemulsion is poured into precooled water at atemperature ranging from 2 to 10° C. under mild stirring, using a wateramount ranging from 10:1 to 80:1 parts by volume with respect to thevolume of the microemulsion. The so obtained dispersion is then washedmany times with distilled water by diafiltration in order to remove thecomponents soluble in water, using a TCF2 equipment(Amicon-Grace-Danvers, USA) equipped with a YM 100 Diaflo membrane witha 100,000 Dalton cut-off, as disclosed in R. Cavalli et al., S. T. P.Pharma Sciences, 2(6), 514-518, 1992.

Such dispersion is finally hot sterilised in autoclave at 121° C. for 15minutes at 2 atm, or freeze-dried.

The so obtained lipidic nanospheres have an average diameter rangingfrom 40 to 300 nm, and preferably from 100 to 200 nm, and apolydispersion index ranging from 0.10 to 0.50.

The characterisation of the microemulsions has been carried out byphotocorrelation spectroscopy with a N 4 Coulter instrument, asdisclosed in R. Cavalli et al., Int. J. Pharm., 148, 47-54, 1997.

Said lipidic substance constitutes the essential active substance of thelipidic nanospheres, which however may include, in particular embodimentforms of the present invention, one or more other pharmacologicallyactive substances.

Such further active substances are typically selected from the groupconsisting of doxorubicin, idarubicin and taxol.

According to a preferred embodiment form of the present invention saidlipidic substance is cholesteryl butyrate.

As surfactants phosphatidylcholine taken from soy or egg yolk,phospholipids and their mixtures are typically used.

According to a preferred embodiment form of the present invention theused surfactant is a commercial product known with the name Epikuron200® (Lukas Meyer, Hamburg, Germany), consisting of phosphatidylcholinefor 95%.

The cosurfactants are selected from alcohols, such as butyl alcohol,carboxylic acids such as butyric and hexanoic acid and bile salts suchas sodium taurocholate and sodium glycocholate.

According to a preferred embodiment form of the present invention, inthe preparation of the microemulsion the various substances are used inthe following proportions, expressed as percentages by weight withrespect to the total weight of the microemulsion:

lipidic substance:  5-18% surfactants: 10-20% cosurfactants: 12-18%water: 44-70%

In the preferred embodiment form the lipidic nanospheres obtained bydiafiltration have a titre in lipidic substance ranging from 25 to 42%,the residue consisting of phosphatidylcholine and/or phospholipids andby traces of other substances used in the preparation process.

According to a particularly preferred embodiment form of the presentinvention the composition of the solid lipidic nanospheres, expressed aspercentage by weight of the various components, is the following one:

cholesteryl butyrate 31.5% phosphatidylcholine 68.0% other 0.5%

Owing to their small size and their composition, said nanospheres havethe unexpected characteristic to be rapidly internalised into cells,where the active substance is fast released.

With respect to the systems of the prior art, said solid lipidicnanospheres therefore are the ideal release system for active substancessuch as the low molecular weight carboxylic acids present in the estersof the present invention. In fact they allow a strong reduction of theeffective doses, with subsequent limitation of the side effects.

The pharmaceutical compositions in form of solid lipidic nanospheres ofthe present invention may therefore be successfully used in thetreatment of all the pathologies for which the above mentionedcharacteristics of fast internalisation of the active substance into thecells are important.

The pharmaceutical compositions in form of lipidic nanospheres object ofthe present invention are useful in the treatment of all the pathologiesfor which the administration of acetic acid, propionic acid, butyricacid and succinic acid is effective, and in particular in the treatmentof the tumoral pathologies and of Mediterranean anaemia.

For said uses the nanospheres according to the invention may be usedalone, or in a mixture with pharmacologically acceptable excipientsand/or diluents, and/or pharmacologically active substances. Inparticular embodiment forms of the pharmaceutical compositions accordingto the present invention, said active substances are antineoplasticagents.

The following examples of preparation of pharmaceutical compositions inform of solid lipidic nanospheres are reported for illustrative, but notlimitative purpose of the present invention.

EXAMPLE 1

a) 15 mg of Epikuron 200® (95% phosphatidylcholine) and 1 mg ofphosphatidylinositol are added to 9 mg of cholesteryl butyrate, and sucha mixture is heated to melting at about 75° C.;

b) a mixture consisting of water (62 mg), sodium glycocholate (3 mg) andbutyl alcohol (10 mg) is heated at the same temperature of the mixtureof the step a);

c) under mild stirring and at the same. temperature of the precedingsteps, the mixture of the step b) is added to the mixture of the stepa), obtaining a microemulsion, which turns out to be clear;

d) the microemulsion obtained in the step c) is dispersed in waterprecooled at 5° C. in an amount equal to 20 parts by volume of water foreach part of microemulsion, obtaining a dispersion of nanospheres;

e) the dispersion obtained in the step d) is washed for 2 times withdistilled water by diafiltration;

f) the washed dispersion is finally freeze-dried.

By photocorrelation spectroscopy the average diameter of the nanosphereshas been determined, which turned out to be equal to 120 nm, with apolydispersion index equal to 0.25.

The so obtained nanospheres consist for 35.5% of cholesteryl butyrateand for 64% of phosphatidylcholine.

EXAMPLE 2

a) 16 mg of Epikuron 200® (95% phosphatidylcholine) are added to 7 mg ofcholesteryl butyrate and heated to the melting of the mixture at about77° C.;

b) at the same temperature of the step a) a mixture consisting of water(62 mg), sodium taurocholate (3 mg) and butyl alcohol (12 mg) isheated;.

c) the mixture of the step b) is added, under mild stirring and alwaysat the same temperature of the previous steps, to the mixture of thestep a), obtaining a clear microemulsion;

d) the microemulsion obtained in the step c) is dispersed in waterprecooled at 2° C. in an amount equal to 40 parts by volume of water foreach part of microemulsion, obtaining a dispersion of nanospheres;

e) the dispersion obtained in the step d) is washed for 3 times withdistilled water by diafiltration;

f) the washed dispersion is finally sterilised according to FU IX at121° C. and at the pressure of 2 atmospheres.

The average diameter of the nanoparticles, determined byphotocorrelation spectrometry, turned out to be 150 nm, with apolydispersion index equal to 0.35.

The so obtained nanospheres consist of 30% cholesteryl butyrate, and of69% phosphatidylcholine.

Tests of Inhibition of the Cell Proliferation

The experimentation has been carried out on NIH-H460 cells of lungcarcinoma (D. N. Carney et al., Cancer Res., 45, 2913-2923, 1985) grownin monolayer in the RPMI 1640 nutrient medium (Bio Whittaker, Verviers,Belgium) added with 10% by volume with respect to the total volume ofFCS (Fetal Calf Serum), at the temperature of 37° C., in a CO₂atmosphere humidified at 5%.

The cells have been put in 24 wells plates, using as nutrient mediumRPMI 1640 added with 10% of FCS, and left to adhere for 24 hours. Theinsemination medium has been then removed and substituted with theexperimental medium consisting of RPMI 1640 with 10% of FCS andincreasing concentrations of sodium butyrate, or of cholesteryl butyratein form of nanospheres prepared as in the above reported example 1. Thecells have been kept for 6 days in contact with such experimentalmedium.

The effect of the sodium butyrate and of cholesteryl butyrate on thecell growth has been estimated counting the cells by a Cell Counter.

Thus it has been observed that the nanospheres containing cholesterylbutyrate induced a complete inhibition of the cell growth at aconcentration equal to 0.21 mM of cholesteryl butyrate, while the sodiumbutyrate, at the same concentration, induced an inhibition of the cellgrowth limited to 50%.

Contemporaneously a comparison test has been carried out usingcholesterol as an additive of the experimental medium consisting of RPMI1640 with 10% FCS, by which it has been observed that the cholesteroldoes not affect the cell proliferation in any way.

The above described experiment has been repeated on cells of themastocarcinoma, identified with the MCF7 abbreviation, using asinsemination medium DMEM/F12 (Dulbecco's modified Eagle's medium, SigmaChemical Co., St. Louis, Mo.) with 2% FCS.

Said cells have been placed in 12 well plates, where they have been leftto adhere for 24 hours in the above described nutrition medium.

The nutrient medium has been then removed and substituted with theexperimental medium consisting of DMEM/F12 with 10% FCS added withincreasing concentrations of sodium butyrate, or of cholesteryl butyratein form of nanospheres prepared according to the above reported example2. The cells have been kept for 6 days in contact with such experimentalmedium.

The antiproliferative effect of the cholesteryl butyrate nanospheres onthe cell growth has been estimated counting the cells with a CellCounter. From such measurements turned out that the nanospherescontaining cholesteryl butyrate induced a complete inhibition of thecell growth at a concentration equal to 0.2 mM of cholesteryl butyrate,while the sodium butyrate induced, at the same concentration, aninhibition of cell growth limited to 40%.

Tests on the Internalization in the Cells

The internalisation of the nanospheres containing cholesteryl butyratein cells of the lung carcinoma, identified with the NIH-H460abbreviation, has been studied by observation at the fluorescencemicroscope.

Operating according to the above reported example 1 nanospherescontaining cholesteryl butyrate have been prepared, which have been madefluorescent by addition of cumarin 6.

NIH-H460 cells, added with 50 μl of tagged nanospheres, have beenincubated at 37° C., and samples have been taken in different times tobe examined.

Said samples have been washed with a saline solution buffered withphosphate buffer, centrifuged and added with a solution containing 5μg/ml of propidium iodide.

The so treated cells have been observed and photographed by fluorescencemicroscope in parallel with the control consisting of the same cellsadded with propidium iodide only.

It has been observed that, contrary to the control, the cells treatedwith the nanospheres made fluorescent by cumarin 6 containingcholesteryl butyrate appeared almost totally fluorescent already after 5minutes from the treatment, demonstrating an almost completeinternalisation of the nanospheres in the cells in very short times.

What is claimed is:
 1. A pharmaceutical composition useful in thetreatment of tumoral pathologies and Mediterranean anaemia in the formof solid lipidic nanospheres having an average diameter lower than 300nm and a polydispersion index ranging from 0.10 to 0.50, wherein saidnanospheres consist essentially of cholesteryl butyrate, the amount ofcholesteryl butyrate in the nanospheres is in the range from 25 to 42%,by weight, and a surfactant selected from the group consisting ofphosphatidylcholine, phospholipids, and mixtures thereof.
 2. Thepharmaceutical composition as claimed in claim 1, wherein the content ofcholesteryl butyrate is 31.5% by weight and the content ofphosphatidylcholine is 68.0% by weight.
 3. A process for the preparationof solid lipidic nanospheres as claimed in claim 1, comprising thefollowing steps: a) heating a mixture comprising a lipidic substance andone or more surfactants at a temperature to take the mixture to themelting; b) heating a mixture consisting of water and one or moreco-surfactants at a temperature equal to the temperature in step a); c)mixing under mild stirring the mixture of step b) with the mixture ofstep a), to form a microemulsion; d) dispersing the microemulsionobtained in step c) in precooled water; e) washing the dispersion ofstep d) with distilled water by diafiltration; f) freeze-drying orsterilizing the product obtained in step e), wherein said lipidicsubstance consists of cholesteryl butyrate and said surfactant isselected from the group consisting of soy phosphatidylcholine, eggphosphatidylcholine, phospholipids and their mixtures.
 4. The process asclaimed in claim 3, wherein the amount of said lipidic substance in saidmicroemulsion of step c) is in the range from 5 to 18 %, by weight, withrespect to total weight.
 5. The process as claimed in claim 3, whereinthe amount of water in the microemulsion of step c) is in the range from44 to 70%, by weight, with respect to the total weight.
 6. The processas claimed in claim 3, wherein the amount of surfactants present in themicroemulsion of step c) is in the range from 10 to 20%, by weight, withrespect to the total weight of the microemulsion.
 7. The process asclaimed in claim 3, wherein the amount of co-surfactants present in themicroemulsion of step c) is in the range from 12 to 18%, by weight, withrespect to the total weight of the microemulsion.
 8. The process asclaimed in claim 3, wherein said dispersion of step d) is carried outwith water cooled to 2 to 10° C. in an amount in the range from 10:1 to80:1 parts by volume with respect to the volume of the mixture ofclaimed step c).
 9. A therapeutic method for the treatment of thetumoral pathologies and Mediterranean anaemia comprising theadministration of an effective amount of the pharmaceutical compositionclaimed in claim 1.